PROJECT ABSTRACT This laboratory's long-term goal is to research the molecular and cellular biology of corneal wound healing. During the current grant period, studies from the lab have significantly improved our understanding of the pathogenesis of diabetic neurotrophic keratopathy (DNK), a major cause of the morbidity of the cornea. We demonstrated that cells in the corneal epithelium communicate through the release of soluble factors, such as TGF?3, IL-1RA, Sema3C (epithelial cells), CNTF (dendritic cells, DC), and VIP (neuron), in response to wounding. Diabetes mellitus disrupts cell-cell interaction, resulting in DNK. Recently, extracellular vesicles, particularly exosomes, were discovered as an additional mechanism for intercellular communication. Exosomes are secreted by most cell types and encapsulate a selective set of proteins, genetic material (for example, mRNAs, miRNAs, lncRNAs, DNA), metabolites, and lipids, and horizontally transfer their cargo to recipient cells. Moreover, these natural nanosized membrane vesicles are excellent delivery vehicles for drugs and therapeutics. We discovered that both cultured mouse corneal epithelial cells (CECs) and bone marrow-derived dendritic cells (BMDCs) release exosomes, which can be readily purified and taken up by each other and by sensory nerves in vitro and/or in vivo. Additionally, we found that the miRNA and protein contents of exosomes vary depending on patho/physiology; that exosomes derived from healing, but not from quiescent CECs, accelerate attenuated wound healing in diabetic mouse corneas; and that exosomes isolated from BMDCs can functionally replace the role of DCs in supporting corneal wound closure. Our published and preliminary studies led to the hypothesis that exosomes communicate critical signaling events in the cornea during wound healing and may be modified to boost therapeutic potential for treating DNK. This application includes three Aims. Aim 1: To explore the molecular mechanism by which CEC exosomes (which have been isolated from quiescent and healing CECs and cultured in normal and high glucose) are therapeutically effective or pathogenic for DNK. Aim 2: To investigate the role of BMDC exosomes (which have been isolated from bone marrow derived DCs) purified from normal and diabetic type 1 and 2 diabetic mice in mediating corneal wound healing and macrophage activation. Aim 3: To determine whether modifying Exo contents can ameliorate DNK as well as impaired skin wound healing in type 1 and 2 mice. Completion of the proposed study will demonstrate exosomes are a significant mediator of cell function or dysfunction depending on the nature of exosomes, identify encapsulated molecules such as proteins and specific microRNAs responsible for exosome regenerative power, and modify purified exosomes to boost their therapeutic potential for treating DNK where there is an unmet clinical need.